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problem with expression of GST fusion protein


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#1 hopfl25

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Posted 27 January 2005 - 03:30 AM

Hi there,

I've got a problem with my protein expression in BL21. The protein I'm investigating normally contains two large inserts which are quite unique. So to study their function I deleted one at a time and both, respectively. I want to express it in BL21 and it works pretty fine except that I get no soluble protein (checked various T and IPTG conc.) - everything is in the pellet. As a control the "native" form with the two inserts is more or less well soluble.

My question now: Does the deletion of one (~75AA) or two (150AA) inserts of the protein have any impact on the correct folding of it?? I mean I disrupt the native form and it seems that all the protein is found in inclusion bodies (misfolded proteins).

Thanks for your comments on that,
Holger

#2 jadefalcon

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Posted 27 January 2005 - 07:34 AM

yes, misfolding could be the reason. one can't really say in advance what will happen if you change a protein in one way ot the other since (as a older collegue of mine told me all the time):

EVERY PROTEIN IS AN INDIVIDUAL!!

and he was (as most of the time) right. so you just have to try and see what will happen. as in your case, you could try to change the conditions of induction like OD at induction, T at growth and expression, concentration of IPTG, method of extraction, etc. It's a long and often futile road, but, who knows, maybe works with your protein... else you could try to resolublise your protein out of the inclusion bodies and refold them....

mike
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